2. Field of the Invention
This invention is related to a method for measuring messenger RNA (mRNA) and polynucleotide-immobilized supports useful in this method. Specifically, this invention is related to measuring methods and reagents in which various types of mRNA can be measured at the same time. In one embodiment of this invention, the mRNA need not be purified from the cell, and the mRNA can be rapidly quantified with high sensitivity.
2. Description of Previous Techniques
Previously well-known techniques for measuring mRNA levels include the Northern Blot Method (See, e.g., J. Sambrook, et al., Molecular Cloning, 2nd ed., Cold spring Harbor Laboratory Press, 1989 hereafter referred to as "Molecular Cloning"), the Dot Blot Method (See, e.g., Molecular Cloning), Ribonuclease Protection Assay (See, e.g., Molecular Cloning), and the Reverse PCR Method (H. A. Erlich (ed.), PCR Technology-Principles and Applications for DNA Amplification, Stockton Press, New York, N.Y., 1989). The disclosures of all of these previous references are hereby incorporated by reference.
The Northern Blot Method detects mRNA which is separated by molecular weight using electrophoresis. The separated molecules are immobilized on a membrane and mRNA levels are determined by hybridization of marker DNA probes. With this method, the molecular weight of the subject mRNA must generally be known. By using this method a complex between a labeled probe, mRNA and an insoluble membrane is formed. The amount of label in the complex at the appropriate site on the membrane is then measured colorimetrically or by radioactive emissions.
The Dot Blot Method detects mRNA by hybridization with a labeled probe after purified mRNA is immobilized as a spot on a membrane. This method can be used to rapidly examine many samples at one time. However, both the Dot Blot and the Northern Blot methods require the use of a specific labeled probe for each mRNA which is desired to be detected.
The Ribonuclease Protection Assay method is used to detect mRNA after it is initially purified. The purified mRNA is hybridized with a labeled RNA probe, and then treated with a ribonuclease that specifically digests single stranded RNA. The labeled RNA probe will form a duplex in the solution and be protected from digestion. This is then immobilized on a membrane after separation of the double-stranded RNA/probe complex.
The reverse PCR method is used where cDNA is synthesized using reverse transcriptase and purified mRNA. This cDNA can be immobilized to a membrane. The synthesized cDNA is then amplified using standard PCR (Polymerase Chain Reaction) methods. After separating cDNA according to its molecular weight by electrophoresis, it becomes a single band. The DNA is then immobilized on a membrane, and detected by hybridization with a labeled DNA probe. However, after PCR, no quantitative information remains due to the amplification of the original message.
All of the foregoing methods take extended periods of time to perform since the mRNA used must be in at least a partially purified form. Also, electrophoresis of the samples and immobilization to a membrane is necessary. Both of these steps reduce the reliability of quantification of the mRNA.
Thus, there remains a need to resolve the above problems, and to provide a method of measuring for rapidly determining messenger RNA levels and a measuring reagent useful in such a method.